This proposal seeks to build upon previous studies of the structure and properties of yeast cytochrome c peroxidase (CCP) and its engineered variants in order to realize our long term goal of introducing novel oxidative reactions into the protein framework. The primary hypothesis underlying these studies is that the potential for the diverse chemical reactivity displayed by heme enzymes resides largely within the heme cofactor, and that a significant role of the protein is to limit or direct access of substrates to this reactive center. In addition, there are many examples where the protein directly modulates the activity of the heme. It has been our goal to delineate the boundaries between these two roles for the protein, and then use this information to introduce new sites where small molecule substrates interact with the heme cofactor, capitalize on the remaining heme-protein interactions, and allow novel reactions to ensue. Studies are proposed along three general lines of inquiry; 1) to generate and study novel intermediates in the reactions of CCP mutants, 2) to develop and extend structure-affinity relationships for small molecule binding to several newly characterized artificial cavities in CCP, and 3) to exploit the knowledge obtained in these studies for the specific oxidation and detection of small molecule substrates. These studies will provide a better understanding of the factors that control the very different activities of the peroxidases on one hand, which oxidize substrates by electron transfer, and monooxygenases on the other, which operate by ferryl mediated oxo-transfer chemistry. They will also provide a novel framework for comparison of the protein-ligand interactions seen in these engineered sites with naturally evolved binding sites, and may reveal insights into enzyme-substrate or ligand-receptor interactions to aid the development of drug design tools.